A variety of techniques is being applied to the investigation of the structure and stability of nucleic acid triplexes. These are of great potential value as chemotherapeutic agents capable of selective gene regulation. This project entails the application of high resolution proton NMR, microcalorimetry, UV/visible spectroscopy and electorphoresis to the experimental determination of the thermodynamics of triplex formation and the molecular details of triplex structure. These results are then utilized to develop theoretical models for these triplex structures. For example, we have recently investigated the triplex formed by adding d(GGGTTTTGGG) to the duplex d(GGGAAAAGGG)- d(CCCTTTTCCC). This work is now being extended to the sequence d(GAGAGAGAGAG) and its Watson-Crick complement. One particularly important and fundamental aspect of these structures is the polarity or orientation of the third strand relative to the purine strand of the duplex. In this case, using fluorescence energy transfer techniques, we have determined that there is a slight preference for one orientation - parallel to the purine strand - relative to the anti-parallel orientation. This implies very little difference in stability of these two structures, and we plan to carry out calculations of stability using AMBER, developed at UCSF, along with visualization and inspection of the resulting structures with MidasPlus.